Interpretive Summary: No method exists to introduce foreign genes into the mitochondria of higher plants. Cucumber possesses three unique attributes that may make mitochondrial transformation possible: paternal transmission, relatively few huge mitochondria in microspores, and the presence of the MSC phenotype. MSC is a paternally transmitted mosaic (MSC) phenotype with weak growth; the leaves of MSC plants are smaller, misshapen, and exhibit a sectoring of green and chlorotic tissues. In this study, we wished to establish the molecular basis of the MSC mutation in cucumber. We identified a major deletion in the mitochondrial DNA of MSC plants, spanning at least 16 kilobases. We demonstrated that this deleted region is transmitted with the MSC phenotype; however, the deleted region carried no expressed genes. The simpliest explanation is that other deletions exist in the mitochondrial DNA and are carried together with the major deletion. This research may lead to a mitochondrial transformation system of higher plants that would speed up the development of cytoplasmic male-sterile lines for hybrid seed production across many crops.

Technical Abstract:
The cucumber mitochondrial genome is unique because of its huge size and paternal transmission. Three cucumber lines derived from two independent tissue culture experiments exhibited a paternally transmitted mosaic (MSC) phenotype. Transmission studies eliminated paternal imprinting, but low levels (less than 1 percent) of wild type testcross progenies were documented. We identified a 16 kb region (JLV5-DEL) in the mitochondrial genome that was deleted in all the three MSC lines. This deletion was transmitted with the MSC phenotype through the F3 and testcross generations. PCR amplification using two primer sets for regions within JLV5-DEL revealed that rare wild type sorters possessed this region, and PCR using mtDNA as the target revealed low levels (less than 0.5 percent) of the wild type region in MSC16. Therefore, MSC plants are heteroplasmic for the deleted region, but at levels below detection by standard PCR and Southern hybridizations using genomic DNA. Sequence analysis revealed no homologies to mitochondrial genes and no open reading frames within JLV5-DEL. Because no genic regions were identified, the specific lesion conditioning the MSC phenotype may be another deletion, mutation, or rearrangement that occurred during tissue culture or in the parental line and is transmitted together with JLV5-DEL within the same mitochondrium.